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Team:Cornell/Week 8
From 2011.igem.org
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July 24th - July 30th
Sunday
Monday
Tuesday, July 26
Lab work done by: Claire Paduano & ?
Lab work done by: Charlie Chung, Nicholas Kramer & Jim Mathew
- Objective:
- To anneal primers containing the Avi-Tag and use as inserts in ligation reactions
- To set up various ligation constructs
- Phosphorylation of Primers
- 5μL 10x T4 DNA Ligase Buffer
- 3μL Primer
- 1μL T4 Polynucleotide Kinase
- 41μL H2O
- 50μL Total
- Incubate at 37°C for 30 minutes
- Heat Inactivation of Polynucleotide Kinase
- Incubate at 65°C for 20 minutes
- Primer Annealing
- Incubate forward and reverse primer in a 1:1 ratio at 95°C for 3 minutes
- Let cool to room temperature for ~10 minutes
- Can also use "Anneal" program on thermocycler
- Ligation
- VioE + Avi-Tag
- 8.8μL pZE12 vector backbone (cut with KpnI and ClaI)
- 0.8μL Primer Pair A
- 0.8μL Primer Pair B
- 4.2μL VioE
Wednesday, July 27
Lab work done by: Youjin Cho, Charlie Chung
- De-salted the ligation samples that were set overnight and transformed them into DH5-alpha electrocompetent cells with ampicillin resistance.
